Preparation and identification of anti-human ICAM-1 scFv / 生物工程学报

To screen the specific anti-human intercellular adhesion molecule-1 (ICAM-1) single chain fragment variable (scFv) using phage display library technology and to identify its biological activity. P1 peptide was used as antigen, and the phage antibodies against human ICAM-1 antigen were panned by four binding-eluting-amplifying cycles using Tomlinson I+J phage display library. After four rounds of selective enrichment screening, the positive clones were determined by PCR, enzyme linked immunosorbent assay (ELISA)-based antigenic cross reaction and Dot blotting. Then the binding specificity and biological activity of purified scFv were identified by Western blotting, competitive ELISA and cell adhesion inhibition assay respectively. Furthermore, four positive clones were first panned through P1 peptide coated-ELISA assay, and then J-A1 was obtained and identified by PCR, ELISA-based antigenic cross reaction and Dot blotting, which could show a specific binding between P1 peptide and human ICAM-1 protein antigen. Subsequently, the purified scFv showed a satisfactory specificity and anti-adhesive activity in competitive ELISA and the cell adhesion inhibition assay. The specific anti-human ICAM-1 scFv was prepared successfully from Tomlinson I+J phage display library, which pave the way for further application of anti-human ICAM-1 scFv for inflammation diseases therapeutics.

Construction and in vitro verification of a new humanized anti-CD19 CAR-T cells with high affinity / 中华血液学杂志

Objective@#To construct humanized anti-CD19 chimeric antigen receptor T cells and investigate its ability to kill leukemia cells in vitro and in vivo. @*Methods@#Humanized anti-human CD19 antibody with a high affinity was obtained based on mouse anti-human CD19 antibody (FMC63). Humanized CD19 CAR-T cells (hCART19) were constructed through transfection of lentivirus carrying a CAR sequence of humanized anti-CD19 scFv into human peripheral CD3+ T cells. The ability of hCART19 to kill leukemia cells and secrete cytokines was detected by LDH release assay and ELISA. The in vivo tumor-killing effect of hCART19 was evaluated in a leukemia mouse model. @*Results@#Several different humanized CD19 single-chain antibodies which were constructed by IMGT database were expressed in the eukaryotic expression vector and purified followed by acquiring humanized CD19 antibody (Clone H3L2) with similar binding ability to FMC63. Humanized CD19 CAR lentivirus vector was constructed and transfected into T cells to obtain hCART19 cells. The LDH release experiment confirmed that the killing rate of target cells was increased gradually along with the increased E/T ratio. When the ratio of E/T was 10∶1, the killing rate of target cells by hCART19 reached a maximum. When Raji cells were used as target cells, the hCART19 cells group had a significantly higher kill rate [(87.56±1.99)%] than the untransduced T cells group [(19.31±1.16)%] and the control virus transduced T cells group [(21.35±1.19)%](P<0.001). ELISA analysis showed that the secretion of IL-2 [ (10.56±0.88) pg/ml] and IFN-γ [ (199.02±12.66) pg/ml] in the hCART19 cells group were significantly higher than those in the untransduced T cells group [IL-2: (3.55±0.26) pg/ml; IFN-γ: (37.63±0.85) pg/ml] and the control virus transduced T cells group [IL-2: (2.92±0.32) pg/ml; IFN-γ: (52.07±3.33) pg/ml](P<0.001). The above experiments also showed similar results when CHO-K1-CD19 cells were used as target cells. Moreover, in a human leukemia xenograft animal model, the results showed that mice in the untransduced T cells group and the control virus transduced T cells group all died within 20 to 30 days, and the hCART19 cell group survived >40 days, which was more than the survival time of the other two groups of mice. The difference was statistically significant (χ2=11.73, P=0.008). @*Conclusion@#Humanized CD19 CAR-T cells with anti-leukemic activity have been successfully constructed, which will lay a foundation for clinical studies in the future.

Construction and in vitro verification of a new humanized anti-CD19 CAR-T cells with high affinity / 中华血液学杂志

Objective: To construct humanized anti-CD19 chimeric antigen receptor T cells and investigate its ability to kill leukemia cells in vitro and in vivo. Methods: Humanized anti-human CD19 antibody with a high affinity was obtained based on mouse anti-human CD19 antibody (FMC63). Humanized CD19 CAR-T cells (hCART19) were constructed through transfection of lentivirus carrying a CAR sequence of humanized anti-CD19 scFv into human peripheral CD3(+) T cells. The ability of hCART19 to kill leukemia cells and secrete cytokines was detected by LDH release assay and ELISA. The in vivo tumor-killing effect of hCART19 was evaluated in a leukemia mouse model. Results: Several different humanized CD19 single-chain antibodies which were constructed by IMGT database were expressed in the eukaryotic expression vector and purified followed by acquiring humanized CD19 antibody (Clone H3L2) with similar binding ability to FMC63. Humanized CD19 CAR lentivirus vector was constructed and transfected into T cells to obtain hCART19 cells. The LDH release experiment confirmed that the killing rate of target cells was increased gradually along with the increased E/T ratio. When the ratio of E/T was 10∶1, the killing rate of target cells by hCART19 reached a maximum. When Raji cells were used as target cells, the hCART19 cells group had a significantly higher kill rate [(87.56±1.99)%] than the untransduced T cells group [(19.31±1.16)%] and the control virus transduced T cells group [(21.35±1.19)%](P<0.001). ELISA analysis showed that the secretion of IL-2 [ (10.56±0.88) pg/ml] and IFN-γ [ (199.02±12.66) pg/ml] in the hCART19 cells group were significantly higher than those in the untransduced T cells group [IL-2: (3.55±0.26) pg/ml; IFN-γ: (37.63±0.85) pg/ml] and the control virus transduced T cells group [IL-2: (2.92±0.32) pg/ml; IFN-γ: (52.07±3.33) pg/ml](P<0.001). The above experiments also showed similar results when CHO-K1-CD19 cells were used as target cells. Moreover, in a human leukemia xenograft animal model, the results showed that mice in the untransduced T cells group and the control virus transduced T cells group all died within 20 to 30 days, and the hCART19 cell group survived >40 days, which was more than the survival time of the other two groups of mice. The difference was statistically significant (χ(2)=11.73, P=0.008). Conclusion: Humanized CD19 CAR-T cells with anti-leukemic activity have been successfully constructed, which will lay a foundation for clinical studies in the future.

Development of methods for quality control of humanized monoclonal antibody agaist vascular endothelial growth factor / 中国药学杂志

OBJECTIVE: To develop methods for quality control of humanized monoclonal antiboy (McAb) against vascular endothelial growth factor (VEGF). METHODS: The purity of humanized anti-VEGF McAb was determined by reduced and non-reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS), while the contents of monomer and polymer were determined by size-exclusive high performance liqud chromatography (SE-HPLC) at the concentration of 0.5 and 25.0 mg·mL-1, respectively. Isoelectric point was determined by isoelectric focusing capillary electrophoresis (cIEF), and the content of charge isomers was controled by cation exchange HPLC(CEX-HPLC). For identity, Lys-C digested peptide map was analyzed by RP-HPLC and LC-MS. The biological potency of anti-VEGF McAb was determined with a human umbilical vein endothelial cells (HUVEC) proliferation inhibitory test. And other quality indexes required by the Ch.P and other relevant requirements were also investigated. RESULTS: The purities of drug substance (DS) and products (DP) determined by reduced CE-SDS were (97.77±0.25)% and (97.43±0.57)%, respectively. The purities of DS and DP determined by non-reduced CE-SDS were (98.97±0.15)% and (98.73±0.06)%, respectively. In the SE-HPLC analysis, when the concentration of McAb was 0.5 mg·mL-1, the monomer contents of DS and DP were (98.07±0.55)% and (98.20±0.52)%, while the contents of polymer in DS and DP were (2.00±0.53)% and (1.93±0.55)% at the concentration of 25.0 mg·mL-1. In the CEX-HPLC analysis, the contents of the acidic, main, and basic components of DS and DP were (18.33±0.64)% and (18.60±0.44)%, (69.03±0.80)% and (69.20±0.44)%, (12.70±1.37)% and (12.20±0.87)%, respectively. In the cIEF and LC-MS tests, the DS and DP showed consistency in isoelectric point and peptide map with reference materials. The other quality indexes all met the requirements in Ch.P and other relevant requirements. CONCLUSION: The methods for quality control of McAb against VEGF is successfully developed, which can be used for the routine quality control of the product.

Determination of MIL50, a humanized antibody, in different tissues of rats by a sandwich enzyme-linked immunosorbent assay / 军事医学

Objective To develop a sandwich enzyme-linked immunosorbent assay ( ELISA) for the determination of a humanized antibody MIL50.Methods RiVax, a mutant of RTA (a chain of ricin) expressed in E.coli, was used as the coating protein.Horseradish peroxidase (HRP) labeled IgG of goats against humans was used to determine MIL 50 captured by RiVax coated on the plate.Results Compared with ricin, RiVax could bind well with MIL50,indicating it could be used as a coating protein to determine MIL50 instead of holotoxin.Using this method, the detective limit of MIL50 in PBST (PBS containing 0.1%Tween 20) could be as low as 0.030 mg/L.The absorbance value of ELISA for MIL50 in the ser-um and homogenate of the liver , spleen, lung, kidney, muscle of rats was lower than that in PBST .Conclusion The sandwich ELISA is a sensitive method for the analysis of distribution of MIL 50 in rat tissues.

Construction and expression of humanized anti-EGFR antibody / 军事医学科学院院刊

Objective:To express rationally engineered antibodies against EGFR and assess their affinity to EGFR and anti-tumor cell migration effect. Methods:L and V_H genes of humanized antibodies against EGFR were designed and synthesized. Genes encoding V_H and C_H were connected and then cloned into a pIRES based bicistronic expression vector. Gene encoding the corresponding L gene was also cloned into the same vector. 293T cells were transfected with the recombinant plasmid and the antibody expression was confirmed by Western blotting. The antibodies were purified by protein A based affinity chromatography. Binding of the humanized antibody to the EGFR was assessed by Surface Plasmon Renainance with Biacore3000, and the biological activity of the humanized antibody was determined by tumor cell invasion test.Results:Three expression vectors were constructed and the humanized anti-EGFR antibodies were expressed and purified successfully. In reducing SDS-PAGE, the antibodies exhibited two bands of approximately 25×10~3and 50×10~3, respectively. Western blot assay showed that the humanized antibodies had recognition specificity to goat-human IgG antiserum. Biacore assay revealed that the humanized antibody C3 binds to EGFR with high affinity(6.13×10~(-10)M). Cell migration test showed that C2,C3 and C5 could suppress growth and migration of tumor cells.Conclusion:Three anti-EGFR humanized antibodies (C2,C3 and C5) have been constructed and expressed successfully, and the C3 antibody retained high affinity for EGFR and showed improved inhibitory effect on tumor cell growth and migration.

Present Status and Perspectives of Antibody Therapy against Cancer / 日本補完代替医療学会誌

The purpose of this article is to review the current status and future perspectives of antibody therapy against cancer. Eight antibody drugs against cancer are now commercially and clinically available for treatment of cancer in the United States and two of them are also available in Japan. Current data suggest that antibodies or their genes against cancer can be used in order to increase the tumor specificity of various new immunotherapeutic or gene therapeutic approaches against cancer, thereby enhancing the tumoricidal effect of each treatment while reducing the side effects.<br>